1. Introduction

The ability of woody plants to regenerate from tissue cultures offers profound implications for ecological restoration, genetic improvement, and sustainable forestry practices. The exploration into the regenerative capacities of such plants, particularly through in vitro tissue culture methods, has unveiled potential pathways for advancing both agricultural and environmental applications. Salix species, among other woody plants, exhibit notable regeneration capabilities, making them ideal candidates for studies focused on genetic transformation systems necessary for enhancing traits like stress tolerance and growth efficiency.

1.1. Research Context and Importance of Woody Plant Regeneration

The regeneration of woody plants encompasses a wide array of processes, from simple wound healing to the complex development of new organs or even whole organisms. Early practices of plant propagation have historically leveraged the natural regenerative abilities of plants. With advancements postulated by Haverlant in 1992, the field of tissue culture has dramatically evolved, particularly with the discovery of auxin and cytokinin, which have significantly influenced the fate of plant development in culture conditions [1]. This progression in tissue culture technology has been particularly instrumental in fostering studies on woody plants like Populus and Salix, where efforts have been made to introduce traits such as herbicide resistance and improved stress responses through genetic transformation.

1.2. Current Advances in Woody Plant Regeneration Techniques

The application of tissue culture techniques in woody plants serves multiple purposes, from rapid propagation of valuable timber species to conservation of endangered species. Various studies have successfully established regeneration systems across a diverse array of woody plants, enhancing our capacity to manipulate and understand their genetic makeup. For instance, significant milestones have been achieved with Populus in terms of herbicide resistance and with Salix matsudana for its genetic transformation systems using somatic embryogenesis.

1.3. Specific Focus on Salix Species

The genus Salix, known for its resilience and adaptability to harsh environments, presents a unique system for studying plant regeneration. Recent research has delved into the regeneration capacities of various Salix species, demonstrating the critical roles of genotype, explant choice, and hormonal regulation in successful tissue culture [2]. These studies underscore the variability in regeneration potential among species and highlight the tailored approaches needed for effective culture systems.

1.4. Challenges and Prospective Solutions

Despite the advancements in regeneration techniques, the process remains challenging due to genetic and environmental factors that significantly affect the outcomes. Issues such as explant browning, low induction rates, and the complex interaction between genetic traits and culture conditions need targeted solutions to enhance regeneration efficiency. Furthermore, the development of robust genetic transformation systems is essential for imparting desirable traits to improve stress resistance and growth characteristics.

2. Experimental Materials and Methods

This section outlines the detailed experimental approaches, including the preparation and sterilization of materials, preparation of media with various plant hormones, and the procedures followed for the aseptic cultivation of Salix linearistipularis explants. The methodologies aim to ensure reproducibility and accuracy in the regeneration experiments conducted [3].

2.1. Materials

Plant Materials: Explants used were internodes devoid of buds, and terminal and lateral buds from Salix linearistipularis plants, which were grown under in vitro aseptic conditions for approximately 40 days prior to experimentation.

Laboratory Equipment: The experiments utilized standard laboratory equipment including an aseptic clean bench, a plant constant temperature culture box, centrifuge, pipettes, electronic balance, cultural bottles and flats, tweezers, and sterilized surgical knives.

2.2. Reagents and Media Preparation

Table 1 provides a comprehensive overview of the chemicals and reagents utilized in the experimental procedures for plant tissue culture and regeneration of Salix linearistipularis. This table includes essential details such as the company providing each reagent, the specific product codes, quantities, and the recommended storage conditions. These reagents play critical roles in preparing the various growth media and hormonal solutions necessary for the experiments [4]. Each reagent’s storage conditions are specified to ensure their stability and effectiveness during use.

Table 1. List of Reagents

2.3. Media Preparation

Different media were prepared for the growth and differentiation of plant explants. All media were autoclaved at 120°C for 20 minutes and stored in a 70°C oven until use, as shown in Table 2.

Table 2. Media Preparation

3. Experimental Methods

3.1. Aseptic Treatment of Plant Material

Plant explants were first washed with detergent, rinsed with tap water, and then subjected to several rinses with distilled water on an aseptic clean bench. Surface sterilization was achieved using 5% sodium hypochlorite followed by repeated rinses in sterile distilled water. Explants were placed in culture tubes containing different types of sterile media [5]. Each type of explant was exposed to varying concentrations of hormonal treatments to assess their effects on callus induction and plant regeneration. Table 3 outlines the specific experimental setups used to assess the regeneration capabilities of Salix linearistipularis explants under various hormonal treatments.

Table 3. Experimental Treatments and Conditions

3.2. Data Collection and Statistical Analysis

The regeneration outcomes were quantitatively measured by calculating the callus induction rate, callus proliferation rate, and adventitious bud induction rate using the following formulas:

\( Callus Induction Rate (\%)= (\frac{Number of explants with callus }{ Total number of explants})× 100 \ \ \ (1) \)

\( Callus Proliferation Rate (\%)= (\frac{Number of explants with significant callus growth}{ Total number of explants} )× 100 \ \ \ (2) \)

\( Adventitious Bud Induction Rate (\%)= (\frac{Number of explants with buds}{Total number of explants} )× 100 \ \ \ (3) \)

The data collected from the experiments were analyzed statistically to identify significant differences in regeneration capabilities across different treatments, thus allowing for an understanding of the optimal conditions for the regeneration of Salix linearistipularis [6].

4. Experimental Results

This section presents the results obtained from the experimental propagation of Salix linearistipularis using different types and concentrations of plant hormones. The effects of these treatments on callus induction and the proliferation rates of terminal and lateral buds were examined comprehensively.

4.1. Plant Propagation Results

4.1.1. Terminal Buds Propagation. The propagation effectiveness of terminal buds was analyzed under four different culture media: MS, WPM, MS supplemented with 0.1 mg/L IBA, and WPM supplemented with 0.1 mg/L IBA. Observations made 35 days post-cultivation indicated significant differences in plant growth metrics such as total plant length, internode length, growth time, and overall plant vitality, as shown in Table 4.

Table 4. Growth Outcomes for Terminal Buds Across Different Media

As illustrated in Figure 1, panels B and D (not shown here), the addition of IBA to the media significantly enhanced the growth outcomes compared to the basic MS and WPM media [7]. Conversely, lateral buds did not exhibit satisfactory regeneration under any of the media tested, indicating their unsuitability for use as primary explants in regeneration experiments under the given conditions.

Figure 1. Plant propagation in different conditions

4.1.2. Lateral Buds Propagation. Despite initial poor performance, modifications in hormone concentrations improved the regeneration outcomes for lateral buds. Specifically, MS medium supplemented with a low concentration cocktail of 6-BA, IBA, and TDZ was tested. Figure 2 demonstrates that after 30-35 days, lateral buds cultivated in this modified medium exhibited growth comparable to that of terminal buds treated with IBA, fulfilling experimental requirements.

Figure 2. Plant propagation through the lateral buds

4.2. Callus Induction and Proliferation

The impact of various hormonal combinations on callus induction and proliferation was thoroughly assessed. The efficacy of these combinations was quantified by the initiation time, proliferation rate, and physical characteristics of the induced callus [8].

4.2.1. Effects of NAA and 6-BA Combinations. The combination of NAA and 6-BA significantly influenced callus induction and proliferation, with optimal results observed at higher concentrations of 6-BA, as shown in Table 5.

Table 5. Induction and Proliferation Rates at Varied Concentrations of NAA and 6-BA

Figure 3. Callus induction and proliferation of the combination s of NAA (0.1 mg/L) and 6 BA(0.1-1→mg /L) A: NAA 0.1mg/L, 6B-A 0.7mg/L; B: NAA 0.1 mg/L, 6-BA 0.8mg/L; C: NAA 0.1mg/L, 6-BA 0.9mg/L; D: NAA 0.1mg/L, 6-BA 1mg/L

Figure 3 reveals that the combination of 0.1 mg/L NAA with 1 mg/L 6-BA resulted in 100% induction and proliferation rates, with callus exhibiting superior freshness and compactness.

5. Conclusion

The study of woody plant regeneration through tissue culture techniques, particularly focusing on Salix species, offers valuable insights into advancing ecological restoration, genetic enhancement, and sustainable forestry practices. By exploring the regenerative capacities of woody plants and employing in vitro tissue culture methods, researchers have identified potential pathways for enhancing agricultural productivity and environmental conservation. The importance of tissue culture in advancing regeneration studies cannot be overstated. Historical practices of plant propagation have evolved significantly, especially with the advent of tissue culture technology, which has been instrumental in manipulating plant development through the use of hormones like auxin and cytokinin. This technological progression has paved the way for genetic transformation systems aimed at improving traits such as stress tolerance and growth efficiency in woody plants like Populus and Salix.

Current advancements in regeneration techniques have enabled rapid propagation of valuable timber species and conservation of endangered plants. Significant milestones have been achieved in plants like Populus and Salix, with efforts focused on introducing traits like herbicide resistance and enhanced stress responses through genetic transformation. Salix species, renowned for their resilience and adaptability to harsh environments, provide an excellent system for studying plant regeneration. Recent research has elucidated the critical roles of genotype, explant choice, and hormonal regulation in successful tissue culture of Salix species. However, challenges persist, including issues like explant browning, low induction rates, and the intricate interplay between genetic traits and culture conditions. Despite these challenges, experimental studies have demonstrated promising results in propagating Salix linearistipularis. Terminal buds treated with IBA exhibited superior growth outcomes compared to untreated buds, while lateral buds showed improved regeneration potential with modified hormone concentrations.